Electrospinning of PLA/pearl powder nanofibrous scaffold for bone tissue engineering

Abstract

Pearl powder is a natural composite of CaCO₃ and organic matter, which is a promising biomaterial for bone tissue engineering. In the present work, pearl powder incorporated polylactic (PLA) nanofibrous scaffold fabricated via electrospinning process to improve the weak biocompatibility and mineralization ability of PLA, was used as support for deposition of hydroxyapatite (HA) and supporting cell culture. Then, the as-prepared scaffolds were well characterized by several assays. Morphology observations showed that pearl powder can homogeneously distribute on the surfaces of nanofibers, and appreciably better hydrophilicity was obtained on a PLA/pearl scaffold than PLA. After a mineralization process, HA particles successfully deposited on the nanofiber, which fully covered the surfaces of PLA/pearl nanofibers, whereas coverage seemed only partial on pristine PLA. An in vitro test illustrated higher cell proliferation and better adhesion morphology of MC3T3s that appeared on the PLA/pearl nanofibrous scaffold. Therefore, electrospinning a PLA/pearl nanofibrous scaffold has superior properties to pristine PLA, and shows potential application in bone tissue engineering.

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1. Introduction

Porous scaffolds were fabricated through various methods, including templating,¹ foaming,² three-dimensional printing,³ electrospinning,⁴ and so on. As a simple and efficient method, electrospinning has been wildly researched to prepare ultrafine fibers from polymer solution under a high voltage, and fiber diameters can reach from 50 nm to 1 μm or thicker.⁵ Compared with traditional fibers, this nanofiber has many advantages such as higher porosity and surface area.⁶ Thus, it has been extensively applied for matter/ion absorption or releasing such as fields of electrode preparation,⁷ sewage treatment,⁸ and tissue engineering.⁹ For biomedical applications, the structure of a nanofibrous mat architecturally mimics the extracellular matrix (ECM),¹⁰ which is beneficial for cell attachment and proliferation, as well as for accelerating drug delivery.

It is well known that requirements for materials employed in the biomedical field are extremely strict. For decades of research, many materials, organic and inorganic, have been investigated for certain applications. As a large branch of organic materials, biodegradable polymers attracted much attention for a long study period; for instance, polylactic (PLA),¹¹ poly(vinyl alcohol) (PVA),¹² and polycaprolactone (PCL).¹³

In addition, inorganic nanoparticle-doped hybrid materials have also been applied for tissue engineering. These materials contributed functions like drug loading and controlled releasing, as well as sustaining stem cells differentiation and osteoblast proliferation. According to numerous research projects in bone regeneration, inorganic nanoparticles, including hydroxyapatite (HA),¹⁴ carbon nanotubes,⁴ and graphene,¹⁵ have been incorporated in an electrospun nanofibrous mat, for improving the low osteoconduction and degradation rate of a polymer matrix.

As the dominating composition of natural bone, HA has been intensely studied during the scaffold fabricating process. Actually, it was demonstrated that HA has an outstanding osteoconduction property; however, due to its brittleness, its employment is limited in the application of tissue repairing as a single component.¹⁶ Therefore, incorporating with polymer materials to construct a hybrid scaffold shows a great importance in tissue engineering, including enhancing mechanical properties and osteoconductivity.

Meanwhile, bone growth factors (BGF) are often introduced into a scaffold to accelerate differentiation of bone marrow mesenchymal stem cells (BMSCs), which will further improve osteoconductivity based on HA.^17,18 Recently, a naturally degradable biomaterial, pearl powder, was found to possess more promising bone regeneration property than HA through in vitro and in vivo studies; this was attributed to the release of BGF from its component, such as bone morphology proteins (BMPs).^19,20 Besides, CaCO₃, the main component of pearl powder, will participate in HA forming in a long run, which could release Ca²⁺ and experience a process of dissolution–binding–precipitation mechanism.^21,22 Moreover, the presence of pearl powder will strengthen the adhesive force between matrix and deposited HA layer, as well as mineralization rate.²³

Considering the above factors, we attempted to disperse pearl powder into a PLA matrix and a PLA/pearl nanofibrous scaffold was prepared by an electrospinning technique. Afterwards, a series of characterizations were applied to measure the hydrophilicity, mineralizing property, cell adhesion, and proliferation of the PLA/pearl hybrid nanofibrous scaffold (Fig. 1). The improved capacities of mineralization and biocompatibility make this scaffold promising for bone tissue engineering.

Fig. 1 Scheme for preparing PLA/pearl nanofibrous scaffolds and their mineralization.

2. Experimental details

2.1 Materials and reagents

Polylactic (PLA, 2 × 10⁵ g mol⁻¹) and pearl powder (average diameter of 100 nm) were purchased from Guanghua Weiye Co., Ltd, Shenzhen, China. and Changshengniao Pearl Biotech Ltd., Zhejiang, China, respectively. Alpha-minimum essential medium (α-MEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin were obtained from Gibco Life Technologies Co., (Grand Island, NY). Alexa Flour@ 488 phalloidin and DAPI were provided from Invitrogen Trading Co., Ltd (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was purchased from Shanghai Yuanxiang Medical Equipment Co., Ltd. All other chemical reagents were of analytical grade and used without further purification; they were purchased from Lingfeng Chemical Reagent Co., Ltd (Shanghai, China).

2.2 Preparation of nanofibrous scaffold

Firstly, pearl powder was pretreated to improve its compatibility in spinning solution as described in our previous work.²⁴ Briefly, pearl powder was dispersed and sonicated in deionized water, then appropriate amounts of polyethylene glycol (PEG) was added and kept stirring. The obtained aqueous solution was frozen overnight and finally freeze-dried to collect the pretreated pearl powder.

PLA was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) with a concentration of 10 wt% by magnetic stirring under 50 °C for 2 h. Then, the required amount of pearl powder was added to the PLA solution to prepare the spinning solution (1 wt%, 2 wt%, and 3 wt%). Later, the prepared scaffolds were marked as EP0, EP1, EP2, and EP3, respectively.

The as-prepared spinning solution was fed into a 5 mL plastic syringe; the diameter of the capillary tip was 0.40 mm. The electrospinning process was carried out under a conductivity difference of 18 kV provided by a variable high voltage power supply and an extrusion rate of 15 μL min⁻¹ controlled by a syringe pump. The nanofibers were collected on a grounded aluminum foil placed on the surface of an adjustable lab jack as the target. The distance between the target and the tip of the syringe needle was 15 cm, ambient temperature and relative humidity were kept at 25 °C and 30%, respectively. The as-prepared mat was dried under vacuum overnight to remove the residual solvents.

2.3 Mineralization of as-prepared scaffold

Simulated body fluid (SBF) solution was prepared as the ref. 25 and applied to measure the mineralization property of various scaffolds. Hydroxyapatite (HA) deposited on the nanofiber after soaking in SBF. Every sample was immersed in SBF with volume of 20 mL under 37 °C for a week; this was changed every day. The mineralized samples were marked HA@EP0, HA@EP1, HA@EP2, and HA@EP3, respectively.

2.4 Characterization

Scanning electron microscopy (SEM, JSM-5600 LV, JEOL, Japan) was used to observe scaffold morphology changes, especially the HA coating layer after soaking. Distribution of pearl powder in nanofibers was observed though a transmission electron microscope (TEM, JEM-2100, Japan) at an operating voltage of 200 kV.

Contact angles (CA) of scaffolds with different pearl content were measured with an automatic video micro contact angle measurement instrument (OCA40Micro, Dataphysics, Germany) with test conditions including water drop volume of 10 μL, humidity of 30% and temperature of 25 °C, to reflect hydrophilic property changes.

Crystallization behavior of PLA and PLA/pearl scaffolds was studied by differential scanning calorimetry (DSC, TA Q2000, USA). Here, an initial heating process to 200 °C at a heating rate of 50 °C min⁻¹ was undertaken, then followed by a cooling procedure to 25 °C at a cooling rate of 50 °C min⁻¹ and a final heating process to 200 °C at a heating rate of 10 °C min⁻¹. The glass transition temperature (T_g), melting temperature (T_m), and heats of melting were obtained.

Mechanical properties were measured with an electronic universal testing machine (Instron 5969, USA) with a cross-head speed of 5 mm min⁻¹. Every sample was prepared at a size of 4 cm × 1 cm. Elongation measurements at the break and tensile strength were determined.

For morphology changes in samples after a mineralization process, SEM was used again to observe deposited particles on nanofibers. Then the deposited particles were scraped off and measured by Fourier Transform infrared (FT-IR, Nicolet-670, Thermo, USA) and X-ray diffraction (XRD, Rigaku Co., Japan) for chemical structure characterization.

For in vitro experiments, MC3T3 cells (the Chinese Academy of Science, Shanghai, China) were used and incubated with α-MEM with 10% FBS, 100 U mL⁻¹ penicillin, and 100 μg mL⁻¹ streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂. Samples were placed in 24-well dishes and sterilized under UV light for 3 h, then washed with phosphate buffer saline (PBS) three times. 2 × 10⁴ cells per well were seeded on samples, and the media was changed every 2 days.

After incubating for a certain time, old media was removed and samples were washed 3 times with PBS. Then, 360 μL of new media with 40 μL of MTT (5 mg mL⁻¹) solution per well was added, then samples were continuously incubated for another 4 h. The solution was removed and 400 μL of dimethyl sulfoxide (DMSO) was added into each well and the culture plate was placed in a shaker for 10 min under dark conditions at 37 °C. Then 100 μL of the supernatant was transferred to a 96-well microplate and absorbance at a wavelength of 570 nm was measured with a microplate reader (MK3, Thermo, USA). The average value was calculated from four parallel samples.

Cell morphology was evaluated by SEM. For SEM observations, the cells were washed with PBS and fixed with 2.5% glutaraldehyde in PBS buffer for 4 h. Then each fixed sample was dehydrated in an ascending series of ethanol – aqueous solution (10%, 30%, 50%, 75%, 80%, 90%, 95%, and 100%) for 10 min and dried under vacuum. Afterwards, the samples were sputter coated with gold and observed by SEM at an accelerating voltage of 15 kV.

For confocal laser scanning microscopy observation (CLSM), cells were washed with PBS and then fixed with 4% glutaraldehyde for 30 min at 4 °C. Afterwards, the cells were washed by PBS twice and soaked in 0.1% Triton X-100 in PBS for 5 min. After washing in PBS again, the fixed cells were stained with Alexa Flour@488 phalloidin solution (165 nM) for 10 min. For nucleus labeling, the cells were washed with PBS and stained with DAPI solution (100 nM) for 10 min. Then the samples were washed with PBS and observed by CLSM.

The values are all presented as mean ± standard deviation. Statistical analysis was carried out through a one-way analysis of variance (one-way ANOVA) and Scheffe's post hoc test. The criteria for statistical significance were *P < 0.05 and **P < 0.01.

3. Results and discussion

3.1 The morphologies observation and contact angle of nanofibrous scaffolds

PLA and PLA/pearl nanofibrous scaffolds were successfully fabricated by an electrospinning technique, and the presence of pearl powder didn't affect the spinning process. Firstly, the morphology of used pearl powder was observed by SEM and shown in Fig. 2 where it exhibited a structure of flaky aragonite. Then the morphologies of PLA and composite nanofibrous scaffolds with different pearl powder content were observed by SEM; the images and diameter distribution (calculated from at least 75 fibers) are shown in Fig. 3. From the SEM images, it can be seen that PLA and composite nanofibers were well fabricated and beadless. Besides, the fiber surface of PLA is very smooth, while it may become rough on PLA/pearl nanofibers. Obviously, the rough-surface of a composite nanofiber is attributed to the addition of pearl powder, which homogeneously distributes on the surface of hybrid nanofibers.²⁶ Moreover, the amount of exposed pearl powder becomes greater with an increase of its concentration. The average fiber diameter of each sample according to distribution results was 944, 948, 957, and 1031 nm, respectively. In addition, the greater average diameter may possibly be attributed to the increase in viscosity of the spinning solution after adding pearl powder.^27,28 Moreover, the rougher surface will be beneficial for larger amounts of pearl to contact the surrounding environment.

Fig. 2 SEM images of pearl powder. (b) is high magnification of (a).

Fig. 3 SEM images and diameter distribution of PLA and PLA/pearl nanofibrous mat.

TEM images shown in Fig. 4 indicate the structure of pearl powder contained PLA nanofibers where pearl powder homogeneously dispersed in nanofibers. The fiber morphology was affected by the appearance of pearl powder in different degrees, especially in EP3. In addition, no obvious agglomeration of pearl powder was observed in nanofibers, and that may be possibly because of the pretreatment with PEG, as well as the low concentration. From the images of SEM and TEM, it is suggested that the position of pearl powder appeared near the surface of composite nanofibers.

Fig. 4 TEM images of EP0 (a), EP1 (b), EP2 (c), and EP3 (d).

Surface hydrophilicity of biomaterials plays an important role in its application. Fig. 5 shows the water contact angle (CA) of PLA and composite scaffolds. The CA of each sample is 127.1, 121.2, 117.0, and 115.5°, respectively, which decreased with increasing pearl content. PLA is a type of highly hydrophobic polymer, which makes it exhibit a large CA, while maintaining decreasing CA of PLA/pearl scaffold is supposed to be the attribute of good hydrophilicity from pearl powder. According to the morphology of nanofibers observed by SEM and TEM, pearl powder could distribute on the fiber surface, which makes it easy to touch water drops, thus exhibiting smaller CA on composite scaffolds.

Fig. 5 Water contact angle of PLA and PLA/pearl nanofibrous mats.

3.2 DSC analysis and mechanical properties of nanofiber scaffolds

Fig. 6 shows the DSC second heating scan curves of nanofibrous mats; the heating rate of the second heating procedure was 10 °C min⁻¹. The shoulder peak appearing at about 57 °C on each curve corresponds to the glass transition temperature (T_g) of PLA, which was hardly changed by addition of pearl powder. An exothermic peak at 122 °C is exhibited on the curve of pristine PLA plus a single endothermic peak at 168 °C according to melting data, which corresponds to the temperature of cold crystallization (T_c) and melting (T_m), respectively. However, both T_c and T_m changed after the addition of pearl powder. The T_c on the curves of samples containing pearl powder appeared at a little lower temperature region around 116 °C compared to that of PLA, which suggested that the existence of pearl powder enhances the cold crystallization ability of PLA. This may be attributed to pearl powder acting as a nucleus in the scaffold and strengthening the nucleation ability of PLA. In addition, double melting peaks at temperatures of 163 °C and 169 °C were observed with curves of samples containing pearl powder, while only single peak appeared on the curve of PLA. In general, multiple melting behaviors are related to the existence of crystals with more than one kind of crystal form, causing the melting-recrystallization-re-melting procedure during a heating process. The polymorphism behavior of PLA, which intensely relies on crystallization conditions, has been studied. Under slower cooling rates, a similar double-melting-peak situation also appears with PLA, which offers a relatively competing crystallization process for neat PLA.²⁹ Whereas in this study, the cooling rate of samples was −50 °C min⁻¹, which could quench PLA and lead to a low degree of crystallinity. Nevertheless, double melting peaks appeared only on composite samples under the same and relatively fast cooling temperatures, which indicated that the crystallization process of PLA was affected by the addition of pearl powder.³⁰

Fig. 6 DSC analysis of PLA and PLA/pearl nanofibrous mat.

Besides, the calculated heats of melting (ΔH_m) of each sample according to the DSC curve is provided in Table 1. The ΔH_m of each sample was 39.13, 40.02, 36.28, and 36.19 J g⁻¹, respectively, indicating that crystallization ability was improved by adding pearl powder, and EP1 exhibited the highest ΔH_m.

Table 1 The ΔH_m of PLA and PLA/pearl nanofibrous mat during melting process

Sample	EP0	EP1	EP2	EP3
ΔHm (J g^−1)	39.13	40.02	36.28	36.19

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Fillers always play dual roles in a polymer matrix, one acting as a nucleating agent to improve the crystallization ability of polymer in a certain content range, and the other one acting as a hindrance to hamper crystallization process of the polymer if it exceeds the limited content, and possibly making defects in the structure.³¹ The tensile strength and elongation at break (Fig. 7) were both raised when pearl concentration was 1 wt%. According to the DSC analysis result, EP1 showed the best crystallization ability, indicating that the nucleating agent effect of pearl powder plays dominating roles relative to hindering behavior, and thus exerted the best mechanical properties. However, this reversed when content of pearl powder reached 2 wt% or higher, revealing EP2 and EP3 exceeded the limited content and resulted in lower mechanical properties.

Fig. 7 Mechanical properties of PLA and PLA/pearl nanofibrous mat.

3.3 Mineralization ability evaluation of nanofibrous scaffold

Simulated body fluid (SBF) was applied to evaluate the mineralization ability of samples, and SEM images of HA@EP0, HA@EP1, HA@EP2, and HA@EP3 are shown in Fig. 8. Obviously, after soaking scaffolds in 1.5 SBF for a week, hydroxyapatite (HA) successfully deposited on fiber surfaces. However, HA fully covered EP1, EP2, and EP3, and appeared like strings of beads, while less HA formed on EP0. High magnification pictures (Fig. 8(e–h)) illustrate that HA particles aligned orderly and compactly on PLA/pearl nanofiber, whereas they showed scattered distribution on pristine PLA fiber. These results indicated that PLA/pearl showed better mineralization ability than PLA, which may be the reason that pearl acts as a nucleus supporting HA deposition.²³

Fig. 8 SEM pictures of mineralized samples: (a) HA@EP0, (b) HA@EP1, (c) HA@EP2, and (d) HA@EP3; (e–h) are high magnifications of (a–d), respectively.

3.4 Chemical composition and structure characterization of deposited hydroxyapatite particles

Next, FT-IR and XRD characterizations were applied on the deposited HA particles. In Fig. 9(a), the FT-IR spectra revealed that HA was a type of calcium-defection. The band of 1656 and a broad band about 3436 cm⁻¹ result from O–H of absorbed water on HA. The peaks around 563, 603, and 1039 cm⁻¹ are derived from the PO₄³⁻ group. The band at 1426–1477 and 873 cm⁻¹ originates from the CO₃²⁻ group. These results indicate that –OH and PO₄³⁻ of HA particles are partially occupied by CO₃²⁻ or other groups. The XRD pattern in Fig. 9(b) shows two significant peaks at 2θ of 26° and 32°, caused by planes (002) and (211) respectively, which is consistent with HA. This may suggest that the crystal structure doesn't obviously change.³²

Fig. 9 FT-IR (a) and XRD (b) characterization of deposited HA particles.

3.5 Proliferation and morphology observation of cells on nanofibrous scaffolds

As a commonly applied method to evaluate cytotoxicity and cell proliferation in vitro, MTT was also used in this study to evaluate biocompatibility of the scaffold. MC3T3s were initially seeded on its surface, then followed by a MTT assay, SEM observation, and confocal laser scanning microscopy. As shown in Fig. 10, the O.D. value of PLA and PLA/pearl nanofibrous scaffolds after incubating for 1, 4, and 7 days was measured using a MTT assay. Obviously, the O.D. value keeps increasing with extending culture time. Generally, EP0 showed the worst biocompatibility at each time point compared to the PLA/pearl scaffold and control group. After culturing for 1 d, PLA/pearl scaffolds exhibited little higher O.D. value than EP0; however, no obvious difference was found among scaffolds with different pearl contents. However, the change trend of MTT value on different samples was more obvious for long culture periods, and this difference of cell proliferation became clear at time point of 4 d and 7 d. The O.D. value of PLA/pearl scaffolds remained higher as before, moreover, scaffold with higher pearl content showed much higher O.D. values. In addition, the gaps between them were getting larger after incubating for longer periods. Thus, the existence of pearl powder didn't show obvious toxicity with cells, and PLA/pearl powder nanofibrous scaffolds could offer a better cell proliferation rate compared to pristine PLA.

Fig. 10 MTT assay of PLA and PLA/pearl scaffolds after cells were seeded for different times (significance difference compared to other groups *P < 0.05 and **P < 0.01).

Tissue engineering scaffolds should not only possess good cell proliferation rates, but also provide superior places to support cell attachment. Thus, cell morphology on PLA and PLA/pearl powder nanofibrous scaffolds was observed through SEM after incubating for 3 d (2 × 10⁴ cells per well) which is shown in Fig. 11. Cells successfully attached on the surface of each sample with extending morphology, and showed no obvious difference between PLA and PLA/pearl powder. It is revealing that pearl powder didn't affect the cell morphology on fiber surfaces. However, cell numbers on PLA were slightly reduced compared to PLA/pearl powder, which corresponded to the result of MTT assay.

Fig. 11 The SEM images of MC3T3s growth on (a) EP0, (b) EP1, (c) EP2, and (d) EP3; (e–h) are high magnifications of (a–d), respectively.

The above conclusion was further proved by confocal laser scanning microscopy (CLSM). The same number of cells were seeded on all samples for the same time as with SEM evaluation. Fig. 12 illustrates the confocal pictures of cells seeded on different samples. Attachment condition of the cells was affected by the nanofibers morphology according to the red fluorescence in the pictures of experiment groups, and cell morphology on PLA/pearl powder scaffolds seems appreciably better than on PLA scaffold in CLSM pictures. Based on the blue fluorescence of the cell nucleus, EP0 shows the lowest viable cell number among all samples. Due to the homogenously distributed pearl powder on nanofibers, better hydrophilicity and cell proliferation ability are shown on PLA/pearl nanofibrous scaffolds. Actually, it does exhibit a positive effect to achieve better cell morphology when incorporated in scaffold.³³ Therefore, this suggests that the existence of pearl powder was beneficial for cell proliferation and adhesion.

Fig. 12 Confocal laser scanning microscopy images of MC3T3 cells seeded on PLA and PLA/pearl nanofiber scaffolds after culturing for 3 d. Scale bar = 75 μm.

4. Conclusion

In summary, a PLA/pearl nanofibrous scaffold was successfully prepared via electrospinning. The average fiber diameter slightly increased when pearl powder content increased, the fiber morphology was smooth and beadless, and pearl powder could homogeneously distribute on the surface of hybrid nanofibers. As a result, a small enhancement of hydrophilicity was obtained on PLA/pearl scaffolds. The mineralization process made HA particles deposit on sample surfaces, which fully covered the PLA/pearl scaffold after several days, while only partially covering a pure PLA scaffold. In addition, in vitro assays including MTT assay, SEM, and CLSM observation revealed that higher cell proliferation and better adhesion morphology appeared on the PLA/pearl scaffold, indicating that the presence of pearl could improve the weak biocompatibility of PLA. Hence, a PLA/pearl composite nanofibrous scaffold would be superior to PLA, which implies great potential applications in bone tissue engineering.

Acknowledgements

This study was supported by the Fundamental Research Funds for the Central Universities (16D310610), Chinese Universities Scientific Fund (CUSF-DH-D-2016040).

Notes and references

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